The in vitro characterisation of prion diseases of sheep
One of the critical challenges in the transmissible spongiform encephalopathy (TSE) field is to understand the molecular basis of prion propagation and decipher the enigma of prion strains and their role in TSEs. The research approach adopted in this dissertation tackled different subjects of keen i...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2012
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| Online Access: | https://eprints.nottingham.ac.uk/12915/ |
| _version_ | 1848791606977626112 |
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| author | Taema, Maged M. |
| author_facet | Taema, Maged M. |
| author_sort | Taema, Maged M. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | One of the critical challenges in the transmissible spongiform encephalopathy (TSE) field is to understand the molecular basis of prion propagation and decipher the enigma of prion strains and their role in TSEs. The research approach adopted in this dissertation tackled different subjects of keen interest for prion characterisation and diagnostics.
A high throughput enzyme-linked immunosorbent assay (ELISA) was developed for detection of disease-related prion protein (PrPSc) using the protease thermolysin. Thermolysin allowed isolation of protease resistant PrPSc in its full-length form, while cellular prion protein (PrPC) was digested. With further extraction and precipitation of PrPSc with sodium phosphotungstic acid (NaPTA) and in conjunction with using monoclonal antibodies that recognise distinct epitopes, PrPSc was detected and quantified successfully.
Molecular strain typing of ruminant TSEs was investigated using Western blotting and depending on the resistance of PrPSc to digestion with proteinase-K (PK) and thermolysin. The methods discriminated clearly between classical ovine scrapie and experimental ovine BSE. In contrast, experimental CH1641-like isolates showed heterogenous molecular profiles. In addition, the findings from this study demonstrated the existence of thermolysin-sensitive PrP isoforms which are resistant to PK and their presence varied between individual sheep and brain regions.
When studying prion propagation using the serial protein misfolding cyclic amplification (sPMCA) technique, different strains/isolates of ruminant prions were successfully amplified in vitro from as little as 0.01 ng of brain seed. Furthermore, ovine BSE was readily amplified in vitro in brain substrates from sheep with homozygous VRQ or AHQ Prnp genotype. In contrast, the CH1641 strain was refractory to such amplification. This method allowed for differentiation of experimental BSE from CH1641 prion strains within an ovine host, providing hope for the potential of sPMCA as a strain typing assay.
The use of bacterially expressed recombinant PrPsen (rPrPsen) as substrate in PMCA reactions (rPrP-PMCA) was assessed. The use of the substrate improved the sensitivity, specificity, practicality and speed of sPMCA assays for detecting a range of ovine prion isolates. Expression and purification of recombinant Syrian hamster prion protein (Sha rPrP) and VRQ ovine PrP (VRQ rPrP) provided substrate for detecting PrPSc in scrapie affected brain samples. Although both substrates had the same level of sensitivity, rSha PrPsen had better specificity than VRQ rPrP. There were variations in amplification efficiency between different batches of the same rPrP.
This study recommends further investigations looking at the use of a range of experimental CH1641 and BSE samples, as well as using panels of CH1641-like field isolates for sPMCA reaction to establish (such) strain typing methodology. Furthermore, applying the rPrP-PMCA assay to detect PrPSc in secreta and excreta of infected sheep in the pre-clinical phase of the disease may provide a non invasive ante-mortem test for scrapie. |
| first_indexed | 2025-11-14T18:31:11Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-12915 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T18:31:11Z |
| publishDate | 2012 |
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| repository_type | Digital Repository |
| spelling | nottingham-129152025-02-28T11:22:05Z https://eprints.nottingham.ac.uk/12915/ The in vitro characterisation of prion diseases of sheep Taema, Maged M. One of the critical challenges in the transmissible spongiform encephalopathy (TSE) field is to understand the molecular basis of prion propagation and decipher the enigma of prion strains and their role in TSEs. The research approach adopted in this dissertation tackled different subjects of keen interest for prion characterisation and diagnostics. A high throughput enzyme-linked immunosorbent assay (ELISA) was developed for detection of disease-related prion protein (PrPSc) using the protease thermolysin. Thermolysin allowed isolation of protease resistant PrPSc in its full-length form, while cellular prion protein (PrPC) was digested. With further extraction and precipitation of PrPSc with sodium phosphotungstic acid (NaPTA) and in conjunction with using monoclonal antibodies that recognise distinct epitopes, PrPSc was detected and quantified successfully. Molecular strain typing of ruminant TSEs was investigated using Western blotting and depending on the resistance of PrPSc to digestion with proteinase-K (PK) and thermolysin. The methods discriminated clearly between classical ovine scrapie and experimental ovine BSE. In contrast, experimental CH1641-like isolates showed heterogenous molecular profiles. In addition, the findings from this study demonstrated the existence of thermolysin-sensitive PrP isoforms which are resistant to PK and their presence varied between individual sheep and brain regions. When studying prion propagation using the serial protein misfolding cyclic amplification (sPMCA) technique, different strains/isolates of ruminant prions were successfully amplified in vitro from as little as 0.01 ng of brain seed. Furthermore, ovine BSE was readily amplified in vitro in brain substrates from sheep with homozygous VRQ or AHQ Prnp genotype. In contrast, the CH1641 strain was refractory to such amplification. This method allowed for differentiation of experimental BSE from CH1641 prion strains within an ovine host, providing hope for the potential of sPMCA as a strain typing assay. The use of bacterially expressed recombinant PrPsen (rPrPsen) as substrate in PMCA reactions (rPrP-PMCA) was assessed. The use of the substrate improved the sensitivity, specificity, practicality and speed of sPMCA assays for detecting a range of ovine prion isolates. Expression and purification of recombinant Syrian hamster prion protein (Sha rPrP) and VRQ ovine PrP (VRQ rPrP) provided substrate for detecting PrPSc in scrapie affected brain samples. Although both substrates had the same level of sensitivity, rSha PrPsen had better specificity than VRQ rPrP. There were variations in amplification efficiency between different batches of the same rPrP. This study recommends further investigations looking at the use of a range of experimental CH1641 and BSE samples, as well as using panels of CH1641-like field isolates for sPMCA reaction to establish (such) strain typing methodology. Furthermore, applying the rPrP-PMCA assay to detect PrPSc in secreta and excreta of infected sheep in the pre-clinical phase of the disease may provide a non invasive ante-mortem test for scrapie. 2012-12-12 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/12915/1/THE_IN_VITRO_CHARACTERISATION_OF_PRION_DISEASES_OF_SHEEP.pdf Taema, Maged M. (2012) The in vitro characterisation of prion diseases of sheep. PhD thesis, University of Nottingham. |
| spellingShingle | Taema, Maged M. The in vitro characterisation of prion diseases of sheep |
| title | The in vitro characterisation of prion diseases of sheep |
| title_full | The in vitro characterisation of prion diseases of sheep |
| title_fullStr | The in vitro characterisation of prion diseases of sheep |
| title_full_unstemmed | The in vitro characterisation of prion diseases of sheep |
| title_short | The in vitro characterisation of prion diseases of sheep |
| title_sort | in vitro characterisation of prion diseases of sheep |
| url | https://eprints.nottingham.ac.uk/12915/ |